The function of the cochlea is to transduce complex sound pressure waves into electrical signals. Organ of Corti vibration is based upon a complex interplay between passive mechanical structures and active OHC- based processes. While laser Doppler vibrometry has added tremendously to our understanding of cochlear physiology, this technique is limited. Only motion from one point on the basilar membrane can be measured, and this provides only a surrogate measure for what is actually responsible for the sense of hearing: deflection of the IHC stereociliary bundle. Thus, these measurements alone cannot explain how the cells and tissues within the organ of Corti work collaboratively to develop cochlear amplification. Vibratory measurements of all of the structures within the intact organ of Corti are needed to understand this process. We have developed a novel technique, volumetric optical coherence tomography vibrometry (VOCTV, pronounced voctive) that overcomes these limitations because it can image directly through the mouse otic capsule bone and simultaneously resolve vibrations at every voxel. Thus, we are at the cusp of understanding how the active and passive mechanics of the cochlea drive IHC stimulation, i.e. the input received by the brain. Our preliminary data demonstrate that frequency-dependent differential motion within the organ of Corti exists. We hypothesize that these movements are mechanically coupled to tilting of the OHC-Deiter cell junction, that the tilting varies with the passive stiffness of the HC, and that the tilting is enhanced by OHC electromotility. This hypothesis is important because, if true, it means that OHC electromotility improves hearing not by increasing vertical displacements of the organ of Corti, but by converting vertical displacement of the basilar membrane into radial fluid movement that can stimulate IHCs. Our studies will explicitly measure the role of the OHC by comparing the in vivo vibratory patterns of wild-type mice (normal OHC stiffness, normal electromotility), prestin 499 mice (normal OHC stiffness, no electromotility), and prestin null mice (decreased OHC stiffness, no electromotility). Aim 1 is to use our existing 1D-VOCTV system to study mice positioned at two different angles to measure both vertical and radial displacements throughout the organ of Corti. Aim 2 is to develop the optical technology and software to perform simultaneous 3D vibratory measurements for every voxel (3D-VOCTV). Aim 3 is use 3D-VOCTV in living mice to measure transverse, radial, and longitudinal motion. If our hypothesis is true, wild- type mice will demonstrate significantly largr radial and/or longitudinal displacements of the OHC-Deiter cell junction compared to dead wild-type, live prestin 499, and live prestin null mice. In addition, the frequency tuning of these displacements should be less sharp in prestin null mice compared to prestin 499 mice. The data obtained with this grant will likely explain the basis for the unique and highly structured anatomy of cells within the organ of Corti. The state-of-the-art technology developed with this grant is likely to become the new standard for making in vivo vibratory measurements.